Document Detail


Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells.
MedLine Citation:
PMID:  15162459     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. However, the intracellular behavior and properties of the enzyme are not well characterized. Specifically, information on the intracellular kinetics and stability of luciferase activity is necessary for real-time luminescence counts from live cells to be quantitatively meaningful. Here, we report a dynamic analysis of luciferase activity in the context of living mammalian cells. We have determined the relative light units measured in living cells to be proportional to that found in cell lysate. We have also calculated the K(m) of luciferase in living cells to be approximately 1 mM, a value much higher than the 10 microM found for pure enzyme in vitro. In addition, a 2-hour half-life of luciferase activity in live cells was measured in real time. Finally, we have modeled luciferase activity in live cells for the purposes of understanding and translating the luciferase signal into a more effective metric of gene expression and cell behavior.
Authors:
Jolene M Ignowski; David V Schaffer
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Biotechnology and bioengineering     Volume:  86     ISSN:  0006-3592     ISO Abbreviation:  Biotechnol. Bioeng.     Publication Date:  2004 Jun 
Date Detail:
Created Date:  2004-05-26     Completed Date:  2005-01-03     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  7502021     Medline TA:  Biotechnol Bioeng     Country:  United States    
Other Details:
Languages:  eng     Pagination:  827-34     Citation Subset:  IM    
Copyright Information:
Copyright 2004 Wiley Periodicals, Inc.
Affiliation:
Department of Chemical Engineering and The Helen Wills Neuroscience Institute, MC 1462, University of California-Berkeley, Berkeley, CA 94720-1462, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
Fireflies / enzymology
Gene Expression Regulation
Genes, Reporter*
Humans
Intracellular Space / ultrastructure
Kinetics
Luciferases / analysis*,  genetics*,  metabolism
Luminescent Measurements*
Models, Theoretical
Chemical
Reg. No./Substance:
EC 1.13.12.-/Luciferases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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